Field of the Invention
The present invention relates to the use of polypeptides having peroxygenase activity for site specific hydroxylation of aliphatic hydrocarbons.
Background
A peroxygenase denoted AaP from the agaric basidiomycete strain Agrocybe aegerita (strain TM-A1) was found to oxidize aryl alcohols and aldehydes. The AaP peroxygenase was purified from A. aegerita TM A1 by several steps of ion chromatography and SDS-PAGE, the molecular weight was determined and the N-terminal 14 amino acid sequence was determined after 2-D electrophoresis but the encoding gene was not isolated (Ullrich et al., 2004, Appl. Env. Microbiol. 70(8): 4575-4581).
WO 2006/034702 discloses methods for the enzymatic hydroxylation of non-activated hydrocarbons, such as, naphtalene, toluol and cyclohexane, using the AaP peroxygenase enzyme of Agrocybe aegerita TM A1. This is also described in Ullrich and Hofrichter, 2005, FEBS Letters 579: 6247-6250.
WO 2008/119780 discloses eight different peroxygenases from Agrocybe aegerita, Coprinopsis cinerea, Laccaria bicolor and Coprinus radians; also shown as SEQ ID NOs: 1-8 in the present application.
DE 103 32 065 A1 discloses methods for the enzymatic preparation of acids from alcohols through the intermediary formation of aldehydes by using the AaP peroxygenase enzyme of Agrocybe aegerita TM A1.
A method was reported for the rapid and selective spectrophotometric direct detection of aromatic hydroxylation by the AaP peroxygenase (Kluge et al., 2007, Appl. Microbiol. Biotechnol. 75: 1473-1478).
It is well-known that a direct regioselective introduction of oxygen functions (oxygenation) into organic molecules constitutes a problem in chemical synthesis. It is particularly difficult to catalyse the selective hydroxylation of aliphatic carbohydrates. The products may be used as important intermediates in a wide variety of different syntheses.
In particular the chemical hydroxylation of alkanes is relatively complex, requires aggressive/toxic chemicals/catalysts and leads to a series of undesired by-products.
It is known that an intracellular enzyme, methane monooxygenase (MMO, EC 14.13.25), oxygenates/hydroxylates the terminal carbon of some hydrocarbons. The MMO enzyme consists of several protein components and is formed by methylotrophic bacteria (e.g. Methylococcus capsulatus); it requires complex electron donors such as NADH or NADPH, auxiliary proteins (flavin reductases, regulator protein) and molecular oxygen (O2). The natural substrate of MMO is methane, which is oxidized to methanol. As a particularly unspecific biocatalyst, MMO oxygenates/hydroxylates, as well as methane, a series of further substrates such as n-alkanes and their derivatives, cycloalkanes, aromatics, carbon monoxide and heterocycles. Utilization of the enzyme in biotechnology is currently not possible, since it is difficult to isolate, like most intracellular enzymes, it is of low stability, and the cosubstrates required are relatively expensive.